This invention is in the field of chemical and biological sensing and biotechnology. This invention relates generally devices and methods useful for amplification and/or sensitive detection of molecules, for example nucleic acids.
Recent advances in nanotechnology and integration of top-down and bottom-up fabrication techniques have enabled the realization of integrated devices with nano-scale sensors for the direct, label-free, and electronic detection of biomolecules. These advances are moving towards the vision of personalized and quantitative molecular medicine. Many disease states and disorders in medicine can be attributed to the aberrations and defects in the DNA molecule of individuals. Hence, sequencing the DNA molecule and sequencing of the entire genome in a cost effective, accurate and rapid manner is desirable.
One of the most promising methods being commercialized is the method of ‘sequencing by synthesis’ where optical detection of each nucleotide added to an existing DNA template molecule that is being polymerized, can be used to obtain the sequence of the starting template.
Recently, the entire genome of a human being, James Watson, was completed within two months by the DNA sequencing company 454 Life Sciences using a novel DNA sequencing approach wherein microfabricated high-density picoliter reactors were used to perform sequencing by synthesis. This method still costs about $1 million, well above the $1,000 per genome target of the National Human Genome Research Institute. Various factors determine the current cost structure of the genome sequence technologies.
The current ‘sequence by synthesis’ approaches consist of the following major components: (i) the original target molecule to be sequenced is digested into smaller fragments, (ii) each smaller fragment is amplified by 30 to 40 cycle PCR to increase their number to have a high signal/noise ratio for detection at a later step, (iii) synthesis of the complementary strands is performed and the addition of each nucleotide is detected using either an optical fluorescence signal generation or conversion of pyrophosphates (PPi) released during DNA polymerization into a chemiluminescence signal (pyrosequencing) with minimum photon count of 10000 from approximately 10 million conjugations, (iv) the information obtained from each original strand (or amplified strands of the same type) is pieced back together (assembly) using sophisticated software and algorithms to obtain the sequence of the original molecule.
Sequencing approaches based on micro to nanoscale technological developments are able to improve sequencing speed and read accuracy as well as reduce costs of sequencing. Wider application of genomics in medicine, from personalized medicine to point-of-care advanced diagnostic and predictive genomic tools, could one day be achieved if such advances reduce the cost of genome sequencing to the $1000 target of the National Human Genome Research Institute.
A variety of microscale sensor devices have been developed for label-free detection of DNA. For example, U.S. Patent Application Publication No. 2006/0197118 discloses an extended gate field effect transistor sensor device. Probe molecules are immobilized on an exposed metal sensor electrode for detection of target molecules, e.g., DNA, to produce a change in an electrical characteristic of the field effect transistor.
U.S. Patent No. 7,385,267 also discloses a field effect sensor device including a functionalized silicon nanowire serving as a gate electrode for the field effect device. The surfaces of the nanowires are selectively functionalized with reactive binding partners, e.g., for probing DNA molecules.
U.S. Patent Application Publication No. 2007/0292855 discloses a device for electrical detection of molecular binding between a probe molecule and a target molecule. Several independently addressable electrodes positioned above the device serve to attract and trap charged molecules above a channel of the device.
U.S. Patent No. 6,203,683 discloses a device for analysis of polyelectrolytes, such as DNA, which includes electrodes for transporting molecules by dielectrophoresis and a trapping electrode attached to a heating element for heating trapped molecules, for example for thermocycling reactions.